OBJECTIVE: To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress. METHODS: Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot. RESULTS: Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01). CONCLUSION Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.
Humans ; Rats ; Female ; Animals ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; Tumor Suppressor Protein p53/genetics* ; Apoptosis Regulatory Proteins ; Acupuncture Therapy ; Primary Ovarian Insufficiency/therapy* ; Apoptosis ; RNA, Messenger ; Oxidative Stress ; Acupuncture Points

BACKGROUND: Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys. METHODS: In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining. RESULTS: 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. CONCLUSION Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.
Humans ; Mice ; Animals ; Transcriptome/genetics* ; Ligands ; Kidney/metabolism* ; Acute Kidney Injury/metabolism* ; Ischemia/metabolism* ; Reperfusion Injury/metabolism* ; Sequence Analysis, RNA ; Adaptor Proteins, Signal Transducing/metabolism* ; Tumor Suppressor Proteins/metabolism*

Objective: To explore the prognostic value of circulating tumor DNA (ctDNA) testing in patients with refractory/relapsed diffuse large B-cell lymphoma (R/R DLBCL) undergoing chimeric antigen receptor T-cell (CAR-T) therapy, and to guide the prevention and subsequent treatment of CAR-T-cell therapy failure. Methods: In this study, 48 patients with R/R DLBCL who received CAR-T-cell therapy at the First Affiliated Hospital of Zhejiang University School of Medicine between December 2017 and March 2022 were included. Furthermore, ctDNA testing of 187 lymphoma-related gene sets was performed on peripheral blood samples obtained before treatment. The patients were divided into complete remission and noncomplete remission groups. The chi-square test and t-test were used to compare group differences, and the Log-rank test was used to compare the differences in survival. Results: Among the patients who did not achieve complete remission after CAR-T-cell therapy for R/R DLBCL, the top ten genes with the highest mutation frequencies were TP53 (41%), TTN (36%), BCR (27%), KMT2D (27%), IGLL5 (23%), KMT2C (23%), MYD88 (23%), BTG2 (18%), MUC16 (18%), and SGK1 (18%). Kaplan-Meier survival analysis revealed that patients with ctDNA mutation genes >10 had poorer overall survival (OS) rate (1-year OS rate: 0 vs 73.8%, P<0.001) and progression-free survival (PFS) rate (1-year PFS rate: 0 vs 51.8%, P=0.011) compared with patients with ctDNA mutation genes ≤10. Moreover, patients with MUC16 mutation positivity before treatment had better OS (2-year OS rate: 56.8% vs 26.7%, P=0.046), whereas patients with BTG2 mutation positivity had poorer OS (1-year OS rate: 0 vs 72.5%, P=0.005) . Conclusion: ctDNA detection can serve as a tool for evaluating the efficacy of CAR-T-cell therapy in patients with R/R DLBCL. The pretreatment gene mutation burden, mutations in MUC16 and BTG2 have potential prognostic value.
Humans ; Prognosis ; Receptors, Chimeric Antigen ; Circulating Tumor DNA/genetics* ; Feasibility Studies ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Lymphoma, Non-Hodgkin ; Mutation ; Cell- and Tissue-Based Therapy ; Retrospective Studies ; Immediate-Early Proteins ; Tumor Suppressor Proteins

This study aimed to explore the mechanism of Zhongfeng Xingnao Decoction(ZFXN) in intervening microcirculatory di-sorders in cerebral hemorrhage by network pharmacology and molecular docking techniques. The information on the components of ZFXN was obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and the predicted targets of chemical components were obtained from PubChem and SwissTargetPrediction. The relevant targets of cerebral hemorrhage and microcirculatory disorders were collected from the GeneCards database, and the common targets of the components and diseases were analyzed by the Database for Annotation, Visualization, and Integrated Discovery(DAVID) for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. Visualization of the correlation network was carried out using Cytoscape software to further screen important chemical components for molecular docking prediction with disease targets. The animal experiment validation was performed using modified neurological severity score(mNSS), enzyme-linked immunosorbent assay(ELISA), quantitative real-time polymerase chain reaction(qRT-PCR), immunofluorescence, and Western blot to detect the effects of ZFXN intervention in mice with cerebral hemorrhage. The results showed that there were 31 chemical components and 856 targets in the four drugs contained in ZFXN, 173 targets for microcirculatory disorders in cerebral hemorrhage, and 57 common targets for diseases and components. The enrichment analysis showed that common targets were mainly involved in biological processes, such as cell proliferation and apoptosis, and signaling pathways, such as tumor pathway, viral infection, phosphoinositide-3-kinase/protein kinase B(PI3K/AKT) signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway. Molecular docking results revealed that the common components β-sitosterol of Rhei Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Ginseng Radix et Rhizoma Rubra showed good docking with proto-oncogene tyrosine-protein kinase(SRC), signal transducer and activator of transcription 3(STAT3), phosphoinositide-3-kinase catalytic alpha polypeptide gene(PIK3CA), recombinant protein tyrosine phosphatase non receptor type 11(PTPN11), AKT1, epidermal growth factor receptor(EGFR), calcium adhesion-associated protein beta 1(CTNNB1), vascular endothelial growth factor A(VEGFA), and tumor protein p53(TP53). Moreover, sennoside E of Rhei Radix et Rhizoma showed good docking with MAPK1. The results revealed that the ZFXN relieved the neural injury in mice with cerebral hemorrhage, decreased the expression of S100 calcium-binding protein B(S100β), neuron specific enolase(NSE), matrix metalloproteinase 9(MMP9), tumor necrosis factor α(TNF-α), interleukin 1β(IL-1β), SRC, EGFR, CTNNB1, VEGFA, TP53, glial fibrillary acidic protein(GFAP), and leukocyte differentiation antigen 86(CD86), and increased the expression of p-PI3K, p-AKT, and zona occludens 1(ZO-1). The results indicate that ZFXN may inhibit neuronal apoptosis and inflammatory response through PI3K/AKT/p53 pathway to protect the blood-brain barrier, thereby slowing down microcirculatory impairment in cerebral hemorrhage.
Animals ; Mice ; Tumor Suppressor Protein p53 ; Proto-Oncogene Proteins c-akt ; Molecular Docking Simulation ; Network Pharmacology ; Vascular Endothelial Growth Factor A ; Microcirculation ; Phosphatidylinositol 3-Kinases/genetics* ; Tumor Necrosis Factor-alpha ; ErbB Receptors ; Cerebral Hemorrhage/drug therapy* ; Neoplasms ; Phosphatidylinositols ; Drugs, Chinese Herbal/pharmacology*

BACKGROUND: Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, and its treatment and diagnosis remain a hot research topic. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is highly expressed in a variety of cancer cells and may be associated with the progression of LUAD. This study aimed to investigate the effect of TPX2 on the malignant progression of LUAD cells and the regulatory mechanisms. METHODS: The expression of gene TPX2 in LUAD tissues from The Cancer Genome Atlas (TCGA) database was analyzed by bioinformatics analysis techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TPX2 and miR-218-5p in human lung normal cell lines and human LUAD cell lines. Western blot was used to detect TPX2 protein expression in cell lines and its effect on the expression of key proteins in the p53 signaling pathway. The relationship between TPX2 and miR-218-5p was predicted using bioinformatics and verified by dual luciferase reporter gene assay. Cell counting kit-8 (CCK-8) assay, cell clone formation, cell scratching, Transwell assay, and flow cytometry were used to detect the effects of miR-218-5p and TPX2 on LUAD cell function. RESULTS: TPX2 was significantly overexpressed in LUAD cells, and knockdown of TPX2 inhibited LUAD cell proliferation, migration, and invasion, promoted apoptosis and induced G2/M phase block, and promoted the expression of key proteins in the p53 signaling pathway. miR-218-5p, an upstream regulator of TPX2, could inhibit its expression. Overexpression of miR-218-5p eliminated the malignant development caused by high expression of TPX2, inhibited the malignant processes of LUAD cells such as proliferation and migration as well as promoted the p53 signaling pathway. CONCLUSIONS miR-218-5p targets and inhibits TPX2 expression and exerts an inhibitory effect on the malignant progression of LUAD cells via p53.
Humans ; Lung Neoplasms/genetics* ; Tumor Suppressor Protein p53/genetics* ; Adenocarcinoma of Lung/genetics* ; Adenocarcinoma/genetics* ; Cell Proliferation/genetics* ; MicroRNAs/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Microtubule-Associated Proteins/genetics* ; Cell Cycle Proteins/genetics*

Lung squamous cell carcinoma (LSCC) accounts for approximately 30% of non-small cell lung cancer (NSCLC) cases and is the second most common histological type of lung cancer. Anaplastic lymphoma kinase (ALK)-positive NSCLC accounts for only 2%-5% of all NSCLC cases, and is almost exclusively detected in patients with lung adenocarcinoma. Thus, ALK testing is not routinely performed in the LSCC population, and the efficacy of such treatment for ALK-rearranged LSCC remains unknown. Echinoderm microtubule associated protein like 4 (EML4)-ALK (V1) and TP53 co-mutations were identified by next generation sequencing (NGS) in this patient with advanced LSCC. On December 3, 2020, Ensatinib was taken orally and the efficacy was evaluated as partial response (PR). The progression-free survival (PFS) was 19 months. When the disease progressed, the medication was changed to Loratinib. To our knowledge, Enshatinib created the longest PFS of ALK-mutant LSCC patients treated with targeted therapy since literature review. Herein, we described one case treated by Enshatinib involving a patient with both EML4-ALK and TP53 positive LSCC, and the relevant literatures were reviewed for discussing the treatment of this rare disease.
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Humans ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Lung Neoplasms/pathology* ; Anaplastic Lymphoma Kinase/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Mutation ; Cytoskeletal Proteins/genetics* ; Lung/pathology* ; Oncogene Proteins, Fusion/genetics* ; Protein Kinase Inhibitors/therapeutic use* ; Tumor Suppressor Protein p53/genetics*

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.
Humans ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; MicroRNAs/metabolism* ; Glioma/genetics* ; Genes, Tumor Suppressor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.
Humans ; Doublecortin-Like Kinases ; Hippo Signaling Pathway ; Verteporfin/pharmacology* ; Cell Line, Tumor ; Protein Serine-Threonine Kinases/metabolism* ; Pancreatic Neoplasms/pathology* ; YAP-Signaling Proteins ; Transcription Factors/metabolism* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Tumor Suppressor Proteins/genetics*

OBJECTIVE: To screen for small molecular compounds with selective inhibitory activity against cutaneous melanoma cells with BAP1 deletion. METHODS: Cutaneous melanoma cells expressing wild-type BAP1 were selected to construct a BAP1 knockout cell model using CRISPR-Cas9 system, and small molecules with selective inhibitory activity against BAP1 knockout cells were screened from a compound library using MTT assay. Rescue experiment was carried out to determine whether the sensitivity of BAP1 knockout cells to the candidate compounds was directly related to BAP1 deletion. The effects of the candidate compounds on cell cycle and apoptosis were detected with flow cytometry, and the protein expressions in the cells were analyzed with Western blotting. RESULTS: The p53 activator RITA from the compound library was shown to selectively inhibit the viability of BAP1 knockout cells. Overexpression of wild-type BAP1 reversed the sensitivity of BAP1 knockout cells to RITA, while overexpression of the mutant BAP1 (C91S) with inactivated ubiquitinase did not produce any rescue effect. Compared with the control cells expressing wild-type BAP1, BAP1 knockout cells were more sensitive to RITA-induced cell cycle arrest and apoptosis (P < 0.0001) and showed an increased expression of p53 protein, which was further increased by RITA treatment (P < 0.0001). CONCLUSION Loss of BAP1 results in the sensitivity of cutaneous melanoma cells to p53 activator RITA. In melanoma cells, the activity of ubiquitinase in BAP1 is directly related to their sensitivity to RITA. An increased expression of p53 protein induced by BAP1 knockout is probably a key reason for RITA sensitivity of melanoma cells, suggesting the potential of RITA as a targeted therapeutic agent for cutaneous melanoma carrying BAP1-inactivating mutations.
Humans ; Melanoma ; Skin Neoplasms ; Tumor Suppressor Protein p53 ; Apoptosis ; Cell Division ; Tumor Suppressor Proteins/genetics* ; Ubiquitin Thiolesterase/genetics*

The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.
Animals ; Drosophila/genetics* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Mammals/metabolism* ; Carrier Proteins ; Tumor Suppressor Proteins/metabolism* ; Drosophila Proteins/genetics*